Detailed information on the intramolecular interactions of protein amino groups has largely been unobtainable except to a limited extent from crystallographic studies. Reductive methylation with NaCNBBH3 and (13C) formaldehyde introduces a specific probe for 13C NMR investigation of protein amino groups (Jentoft and Dearborn, 1979, J. Biol. Chem., 254:4359 and Jentoft, et al., 1979, J. Biol. Chem., 254:4366). We have shown that the chemical shifts of 13C-enriched dimethylamino groups are sensitive to the environment and degree of protonation of the amino group, and we have demonstrated that the pKa of dimethylamino groups are very similar to those of unmodified amino groups. Methylated ribonuclease has been studied to elucidate the role of lysine 41 in the active site. The 13C chemical shift and pKa of dimethylated lysine 41 are affected differently by various active site ligands. In addition, interaction of lysine 41 with another residue with a pKa of about 6.5 (in the absence of ligands) was detected. Methylated HEW lysozyme was studied to determine if salt bridges and hydrogen bonds could form and be detected in methylated amino groups. Six of the seven lysozyme amino groups are involved in intramolecular interactions in the crystal, and our results suggest that, in solution, methylated amino groups maintain these interactions. In low salt, each of the 6 methylated lysyl amino groups and the methylated N-terminal amino group give a distinct 13C NMR signal, each group titrates uniquely, and each residue has a different T1 value. Two amino groups are involved in very strong interactions as indicated by their perturbed chemical shifts and abnormal pH dependencies. These studies have established a variety of interactions and properties of protein lysyl and N-terminal amino groups, information that has previously been experimentally unobtainable.